Treatment of human immunodeficiency virus type 1 (HIV) infection with antiretroviral agents invariably leads to selection and accumulation of drug-resistant mutants that arise due to the intrinsically low fidelity of HIV polymerase. Early detection of these growing variants may be clinically relevant. Before the level of clinical importance can be ascertained, technologies that reliably detect small genetic changes in a large sea of closely related viral quasi-species need to be available. We have developed a novel, closed-tube, single vessel RT-PCR method to detect and quantitate mixed populations of wild-type and mutant viral sequences. Our method, called GENE-CODE, uses two separate fluorophore-labeled forward primers and one common reverse primer to analyze mixed populations of closely related genetic materials. Incorporation of a quencher-labeled nucleotide results in a decrease in reaction fluorescence in proportion to the accumulation of PCR product. Fluorescence measurements are performed during extension phase of the PCR to determine the cycle threshold (Ct) in multiple channels. The percent composition of an unknown sample can be determined by measuring the difference between the Ct of the two channels (defined as deltaCt) and plotting that deltaCt onto a previously established standard curve. The standard curve is generated by plotting percent or ratio of one target to the other versus deltaCt for a set of known standard samples. The mixed population test system analyzes the percentage of wild-type to mutant RNA. The data show a large change in deltaCt with a dynamic range from 100% to 0.01% drug-resistant mutant sequence in a standard mixture. We can detect one copy of mutant per 10,000 wild-type RNA copies in a reaction containing 1.5 x 10/7 total target molecules. This method also determines percent make-up of the analyte. We will optimize our system to detect and quantitate 17 antiretroviral resistant HIV mutations. These mutations confer resistance to the three types of commonly proscribed AIDS therapeutics : nucleoside inhibitors of reverse transcriptase (RT), non-nucleoside RT inhibitors and protease inhibitors. Development of this technology to include a larger number of HIV variants could lead to a simple-to-run and widely-used method for research and clinical diagnostics. In the future, scientists may more easily detect and evaluate drug-resistant HIV variants just as they emerge within patient or experimental samples.